Bottom Line:
Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect.Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2.Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

ABSTRACTSmoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

pone-0063353-g001: Identification of Miz1 as a positive regulator of Hh signaling.(A) Illustration of Miz1 mutants. Wildtype (wt) Miz1 (total 803 amino acid residues) contains a POZ domain (amino acid residue 1–104) and a Myc-interacting domain (amino acid residue 641–715) POZ domain is deleted in Miz1ΔPOZ mutant, and the Myc-interacting domain is deleted in Miz1Δ641–715 mutant. (B) Miz1 interacts with Smo, and such interaction is independent of the POZ domain of Miz1. HEK293 cells were transiently transfected with Myc-tagged Miz1 or a POZ domain deletion mutant Miz1ΔPOZ along with Flag-Smo as indicated. Co-immunoprecipitation experiments were performed using the Flag antibody. The presence of Miz1 full length and Miz1ΔPOZ was detected using the Myc antibody. (C) Miz1 also interacts with Gli2, and the POZ domain of Miz1 is required for the interaction between Miz1 and Gli2. HEK293 cells were transiently transfected with HA-Gli2 together with vector, Flag-Miz1, or Flag- Miz1ΔPOZ as indicated. Co-immunoprecipitation experiments were performed using the Flag antibody. The presence of Gli2 was detected using the HA antibody. (D) Miz1 promotes the activation of Gli activity. C3H10T1/2 cells were transfected with the Hh-responsive reporter (9× Gli-luciferase) together with one of the corresponding constructs as indicated. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in cells transfected with pcDNA3 was normalized to 1 and used as control, and the relative Gli activity was obtained by comparing to the control. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, ns: not significant, compared to the pcDNA3 sample, t test). (F) Miz1 activates Gli activity downstream of Smo receptor. Smo−/− MEF cells were transfected with the Hh-responsive reporter (9×Gli-luciferase) and pRL-TK together with one of the corresponding constructs as indicated. Gli reporter activities were determined as described in (D). Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, ns: not significant, compared to the pcDNA3 sample, t test).

Mentions:
The C-terminus of Drosophila Smo receptor contains multiple phosphorylation sites, and the phosphorylation of the C-terminus has been implicated in controlling the activation of Hh signaling in invertebrates [3], [26]. However, the C-terminus of Smo diverges significantly in vertebrates suggesting that vertebrate Smo receptors are regulated differently [26]. In order to identify potential regulators of Hh signaling that interact with mammalian Smo, we performed a yeast-two-hybrid screen against a human brain cDNA library using the last 249 amino acids of the Smo receptor as the bait. Several positive clones were identified to encode the C-terminal portion of Miz1. To confirm the interaction between full-length Smo and Miz1, Flag-tagged Smo (Flag-Smo) was co-transfected with Myc-tagged Miz1 (Myc-Miz1) or a POZ domain deletion mutant Miz1 (Myc-Miz1ΔPOZ) into HEK293 cells. Both the full-length and ΔPOZ Miz1 co-immunoprecipitated with Smo (Figure 1A and 1B), indicating that Miz1 interacts with Smo, and the POZ domain of Miz1 is not required for such interaction.

pone-0063353-g001: Identification of Miz1 as a positive regulator of Hh signaling.(A) Illustration of Miz1 mutants. Wildtype (wt) Miz1 (total 803 amino acid residues) contains a POZ domain (amino acid residue 1–104) and a Myc-interacting domain (amino acid residue 641–715) POZ domain is deleted in Miz1ΔPOZ mutant, and the Myc-interacting domain is deleted in Miz1Δ641–715 mutant. (B) Miz1 interacts with Smo, and such interaction is independent of the POZ domain of Miz1. HEK293 cells were transiently transfected with Myc-tagged Miz1 or a POZ domain deletion mutant Miz1ΔPOZ along with Flag-Smo as indicated. Co-immunoprecipitation experiments were performed using the Flag antibody. The presence of Miz1 full length and Miz1ΔPOZ was detected using the Myc antibody. (C) Miz1 also interacts with Gli2, and the POZ domain of Miz1 is required for the interaction between Miz1 and Gli2. HEK293 cells were transiently transfected with HA-Gli2 together with vector, Flag-Miz1, or Flag- Miz1ΔPOZ as indicated. Co-immunoprecipitation experiments were performed using the Flag antibody. The presence of Gli2 was detected using the HA antibody. (D) Miz1 promotes the activation of Gli activity. C3H10T1/2 cells were transfected with the Hh-responsive reporter (9× Gli-luciferase) together with one of the corresponding constructs as indicated. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in cells transfected with pcDNA3 was normalized to 1 and used as control, and the relative Gli activity was obtained by comparing to the control. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, ns: not significant, compared to the pcDNA3 sample, t test). (F) Miz1 activates Gli activity downstream of Smo receptor. Smo−/− MEF cells were transfected with the Hh-responsive reporter (9×Gli-luciferase) and pRL-TK together with one of the corresponding constructs as indicated. Gli reporter activities were determined as described in (D). Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, ns: not significant, compared to the pcDNA3 sample, t test).

Mentions:
The C-terminus of Drosophila Smo receptor contains multiple phosphorylation sites, and the phosphorylation of the C-terminus has been implicated in controlling the activation of Hh signaling in invertebrates [3], [26]. However, the C-terminus of Smo diverges significantly in vertebrates suggesting that vertebrate Smo receptors are regulated differently [26]. In order to identify potential regulators of Hh signaling that interact with mammalian Smo, we performed a yeast-two-hybrid screen against a human brain cDNA library using the last 249 amino acids of the Smo receptor as the bait. Several positive clones were identified to encode the C-terminal portion of Miz1. To confirm the interaction between full-length Smo and Miz1, Flag-tagged Smo (Flag-Smo) was co-transfected with Myc-tagged Miz1 (Myc-Miz1) or a POZ domain deletion mutant Miz1 (Myc-Miz1ΔPOZ) into HEK293 cells. Both the full-length and ΔPOZ Miz1 co-immunoprecipitated with Smo (Figure 1A and 1B), indicating that Miz1 interacts with Smo, and the POZ domain of Miz1 is not required for such interaction.

Bottom Line:
Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect.Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2.Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

ABSTRACTSmoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.